“This is not really what we are expecting when we are working with affinity mature B cells; we are expecting the reactivity to narrow down to single reactivity,” Dr. Grönwall said.
In a different approach using a large array with over 50,000 peptides, Dr. Grönwall said the extent of this multi-reactivity can be seen with binding to hundreds and even thousands of peptides. “We do find some of our usual suspects, such as filaggrin and collagen type II, but we also see a lot of other antigens targeted by the different monoclonal antibodies. This works by way of the ACPAs recognizing very small, linear, citrullinated-epitopes (binding motifs), as demonstrated by Sahlström et al., some including glycines, and with variations in the mAbs’ binding preferences,” she explained.5
The takeaway from these findings is that the antibodies bind to very small binding motifs, and all have unique signatures in what they bind to.
Emerging evidence from Dr. Grönwall’s lab and others shows citrullinated protein is one of several post-translational modifications targeted by multi-reactive autoantibodies in RA. For example, when using an array similar to the citrulline array, with almost 50,000 peptides that are now carbamylated peptides, a number of monoclonal antibodies have multi-reactivity to carbamylated and acetylated peptides. She stressed that the term multi-reactivity is preferred to cross-reactivity, because “cross-reactivity makes you think that the citrulline is the strongest reactivity, but this is not necessarily always the case.” For example, in a competition experiment conducted in Dr. Grönwall’s lab, the highest affinity for the monoclonal antibody is to acetylated peptides.
Given this multi-reactivity with clones reactive beyond citrullination, Dr. Grönwall said the term anti-modified protein autoantibodies is being used to demonstrate multi-reactivity beyond citrulline-only reactive ACPAs.
She also noted that different ACPA clones have different pathogenic functionality. For example, research by Sun et al. showed that some clones have the ability to mediate fibroblast migration and other clones have the ability to increase osteoclastogenesis.6
“We see these different clones have different reactivity patterns. We think this is important, but so far we don’t know exactly what the targets are and what is defining to have one functionality or another,” Dr. Grönwall said.
Moving back to serology and looking at reactivity in RA patients, unpublished research from Dr. Grönwall’s lab showed higher reactivity toward carbamylation and acylation, particularly in CCP-positive patients. Also, overlap occurs between these reactivities, with carbamylation reactivity primarily seen within the citrulline reactivity, and acetylation reactivity largely seen in patients who have both citrullination and carbamylation reactivity. “We can also see that the carbamylation and acylation activity is primarily seen in RA patients with very high citrullination reactivity, so there seems to be a correlation there,” she noted.
